A. Day C57BL/6 (B6) n=9 B6 + Abx (12 weeks) n=9 B. Supplemental Fig. 1 day 1 I/R 4min Sacrifice C. Number of colonies in culture media B6 Escherichia coli Lactbacillus spp. Lactobacillus murinus Enterococcus fecalis Staphylococcus (MSSA) Bacteroides spp. 1X15 1X16 1X17 B6 + Abx (12 weeks) Escherichia coli Lactbacillus spp. Lactobacillus murinus Enterococcus fecalis Staphylococcus (MSSA) Bacteroides spp. 1X18 (cfu/g) B6 1X15 1X16 1X17 1X18 (cfu/g) pre day1 B6 + Abx B6 D B6 + Abx Grade * Grade Grade * Intraluminal debris BUN Havcr1 μg/dl Brush border region X4 U-Alb / U-Cr.8 Cr μg/dl *P <.5 Necrosis X1 X4 μg/mgcr X1 B6 I/R B6 + Abx I/R Relative gene expression in kidney tissue E.
Supplemental Figure 1. Gut microbiota againsts from tubular injury in a mouse model of AKI. (A) The protocol of antibiotic treatment and I/R induction in C57BL/6 (B6) mice. Prior to I/R, WT mice were treated with 1 g/l ampicillin sodium salt, 1 g/l neomycin sulfate, 1 g/l metronidazole,.5 g/l vancomycin hydrochloride, and.5 g/l gentamicin sulfate in drinking water for 12 weeks prior to I/R. (B) The gut microbiota was reduced to below 1 1 5 colony-forming units (cfu)/g after treatment with antibiotics. (C) The administration of antibiotics led to an enlargement of the cecum in B6 mice. (D, E) The antibiotictreated mice showed more severe tubular injury, as reflected in the acute tubular necrosis (ATN) score, compared with the untreated mice on day 1 after I/R. Statistical analysis was performed using Student s t test. *P <.5. Abx, antibiotic treatment.
A. Feces (Gut) Total amino acid Supplemental Fig. 2 18 16 14 12 1 8 6 4 2 B. Plasma 16 14 12 1 8 6 4 2 C. Kidney 18 16 14 12 1 8 6 4 2 D. Urine 16 14 12 1 8 6 4 2 (nmol/ml) (nmol/ml) all-ilc all-ilc all-ilc all-ilc B6 B6 Sham day 2 B6 I/R day 2 B6 Sham day 1 B6I/R day 1 n = 4-6/group
Supplemental Figure 2. The profile of all amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in the AKI mice. All amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days, 2, and 1. Data are shown as means SEM., undetectable.
A. Faeces (Gut) 16 14 12 1 8 6 4 2 16 14 12 1 8 6 4 2 B. Plasma C. Kidney (nmol/ml) L-amino acid all-ilc all-ilc B6 B6 Sham day 2 B6 I/R day 2 B6 Sham day 1 B6I/R day 1 n= 4-6/group Supplemental Fig. 3 18 16 14 12 1 8 6 4 2 D. Urine 16 14 12 1 8 6 4 2 (nmol/ml) all-ilc all-ilc
Supplemental Figure 3. The profile of L-amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in the AKI mice. The L-amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days, 2, and 1. Data are shown as means SEM., undetectable.
A. Feces (Gut) 12 1 8 6 D-amino acid Supplemental Fig. 4 B6 B6 Sham day 2 B6 I/R day 2 B6 Sham day 1 B6I/R day 1 n= 4-6/group 4 2 12 1 8 6 4 2 B. Plasma C. Kidney (nmol/ml) all-ilc all-ilc 18 16 14 12 1 8 6 4 2 2 18 16 14 12 1 8 6 4 2 D. Urine (nmol/ml) all-ilc all-ilc
Supplemental Figure 4. The profile of D-amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in AKI mice. The D-amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days, 2, and 1. Data are shown as means SEM., undetectable.
A. Feces (Gut) 1.2 1.8.6 D-amino acid/l-amino acid Supplemental Fig. 5 B6 B6 Sham day 2 B6 I/R day 2 B6 Sham day 1 B6I/R day 1 n= 4-6/group.4.2 B. Plasma.12.1.8.6.4.2 C. Kidney.6.5.4.3 all-ilc all-ilc.2.1 D. Urine 1.8 1.6 1.4 1.2 1.8.6.4.2 all-ilc all-ilc
Supplemental Figure 5. The profile of amino acids analyzed with 2D HPLC. We performed metabolomic analysis of the amino acids in in germ-free mice with AKI. The ratios of the D-/ L-amino acids were evaluated in the feces (A), plasma (B), kidney (C), and urine (D) of the mice with/without I/R on days, 2, and 1. Data are shown as means SEM., undetectable.
A. Total amino acid 6 5 4 3 2 1 B. L-amino acid 6 5 4 3 2 1 C. D-amino acid 7 6 5 4 3 2 1 D. D/L amino acid.16.14.12.1.8.6.4.2 Feces (Gut) Supplemental Fig. 6 all-ilc all-ilc all-ilc all-ilc Gf B6 n = 6 Gf B6 I/R 1day n = 5
Supplemental Figure 6. The profile of amino acids analyzed with 2D HPLC in germ-free mice. We performed metabolomic analysis of the amino acids in the feces of germ-free mice. All amino acids (A), L-amino acids (B), D-amino acids (C) and the ratio of the D-/L-amino acids (D) were evaluated in the germfree mice pre-i/r and on day 1 after I/R. Data are shown as means SEM., undetectable.
Supplemental Fig. 7 NS ml/mouse/6 days Water n = 6 2 mm D- n = 6 Water 2 mm D- Water 2 mm D- meas 29.5 29.71 SD 8.9 4.39 TTEST.96 Supplemental Figure 7. The volume of water intake was similar between the untreated mice and the D-serinetreated mice. Statistical analysis was performed using Student s t test. NS, not significant.
Supplemental Fig. 8 A U-ALB/U-Cr *** μg / mgcr Water n = 8 8 mm D-ine n = 6 **P <.1 ***P <.1 Water day1 after I/R 8mM D-ine B *** *** *** ** **** O.D. n = 6/group 1μM 1μM 1μM 1mM 1mM 1mM D- Supplemental Figure 8. Administration of high-dose D-serine augmented kidney injury. (A) Urine albumin excretion increased after high-dose administration of D-serine. (B) Post-hypoxic tubular proliferation was inhibited by D-serine at a dose of 1 mm. Statistical analysis was performed using Student s t test compare to control. *P <.5.
Marker Pre I/R Post I/R 1 2 3 4 5 1 2 3 4 5 Positive control Negative control Supplemental Fig. 9 Supplemental Figure 9. Confirmation of the status of the germfree mice by 16S rdna PCR before and after I/R. No PCR product was detected in the feces of the germ-free mice before and after I/R.